In 1986-1988, a bacterial disease characterized by opaque intestine or intestinal necrosis with high mortality occurred in larval Japanese flounder (Paralichthys olivaceus) reared at private hatcheries and Prefectural Fisheries Experimental Station in Hiroshima Prefecture. From the intestine of the diseased larvae, a Vibrio species was isolated and tentatively named Vibrio sp. INFL (Intestinal necrosis of flounder larvae) group. The disease condition was reproduced by oral administration of the isolate incorporated into live food organisms : rotifer and brine shrimp. Based on the identical morphological and biochemical characteristics of the causative agent and the same disease symptom, this disease proved to be the same disease as the one reported previously in Wakayama Prefecture under the name of “Chohkanhakudakushoh” (disease with opaque intestine), although the serotype of the isolates was not identical.

Chemical analysis and toxic ty assay of Pasteurella piscicida lipopolysaccharide (LPS) preparations used for immunization of yellowtail (Seriola quinqueradiata) were performed. LPS fractions were obtained by the phenol-water (P-LPS) or phenol-chloroform-petroleum ether (PCP-LPS) methods followed by purification with Cetavlon or ultracentrifugation, respectively. P-LPS was composed of 0.87% protein, 24.0% sugar and 36.0% fatty acid. Whereas PCP-LPS of 0.34% protein, 18.0% sugar and 34.2% fatty acid. The monosaccharide of P-LPS and PCP-LPS was respectively : 30.6% and 16.5% hexose; 3.8% and 2.1% heptose; 2.2% and 3.6% pentose; 2.2% and 3.2% 6-deoxyhexose; 3.2% and 2.6% 2-keto-3-deoxyoctonate (KDO); and 2.8% and 2.4% hexosamine. The fatty acids were analyzed by gas-liquid chromatography (GLC) as their methyl esters. Their identities were confirmed by mass spectrometry. Those analyses showed that the principal constituents were : lauric acid, 3-hydroxy lauric acid, myristic acid and palmitic acid. The acute toxicity assay using mice injected i.p. showed that the P-LPS preparation was more toxic than PCP-LPS. The Limulus amoebocyte assay did not show differences between the two preparations.

The lethal toxicity and immunogenicity of salmolysin purified from the culture supernatant of Aeromonas salmonicida were investigated. Median lethal doses of salmolysin to juvenile white-spotted char, Salvelinus leucomaenis by intramuscular or intraperitoneal injection were estimated as 196 and more than 556 μg/kg body weight, respectively. The toxin was detoxified by either heating at 100°C for 10 min, or incubated at 37°C for a week with 0.4% formalin. No lethalities were found when the treated salmolysins were injected intramuscularly into juvenile white-spotted char. Antigenicity of each detoxified toxin was detected by immunodiffusion analysis as these formed a precipitating line against rabbit anti-salmolysin serum. Immunogenicity of heated, formalinized or native salmolysin to adult white-spotted char were found. Agglutinating antibody titers of the fishes immunized with the detoxified or native toxin were about 16 to 64 at three to five weeks after. These results suggest that the detoxified salmolysin may be an effective toxoid to protect the fish from furunculosis.

A panel of monoclonal antibodies (MAb) was used in an immunodot assay to investigate the antigenic relationships of a variety of aquatic birnaviruses from Asia. All of the Asian birnaviruses tested shared a serogroup-specific epitope with related viruses from North America and Europe. Six viruses from Taiwan were identical in MAb reaction patterns to European eel virus (EEV) and differed from the European Ab virus only by the presence of a single epitope. A single isolate from Thailand was identical to the European Sp virus. Ten viruses from Taiwan exhibited a variety of MAb reaction patterns that were distinct from those previously reported for the nine known serotypes of North America and Europe.

The therapeutic efficacy of a 1 : 3 combination of ormetoprim-sulfamonomethoxine (OMPSMM), oxolinic acid (OA), and miloxacin (MLX) was evaluated against drug resistant strains of Edwardsiella tarda. These resistant strains carried on R plasmid encoded with resistance to chloramphenicol, tetracycline, and sulfonamide. Fish were injected intraperitoneally with the bacterium and orally dosed with various level of OMP-SMM, OA, and MLX for five days. Appropriate positive and negative controls were incoporated into the study. A dose of at least 25 mg/kg/day of a combination of OMP-SMM, 12.5 mg of OA, and 6.2 mg of MLX provided protection. Our results indicated that OMP-SMM, OA, and MLX were effective against the infection with drug resistant strain of E. tarda.

Protective immunity against Streptococcal disease was investigated in rainbow trout, Oncorhynchus mykiss by immersion or intraperitoneal injection of a formalin-killed bacterin. Although no agglutinating antibodies were detected in the serum of fish vaccinated by the immersion method, antibodies were detected in the serum of fish vaccinated by intraperitoneal injection. Bactericidal activities of the serum of fish vaccinated by either the immersion or injection method did not increase. The phagocytic activities of the kidney increased in the vaccinated fish. When the vaccinated fish were challenged with the live pathogen, the number of streptococcal cells in the kidney, spleen, liver, and blood decreased gradually and, in most organs, streptococcal cells disappeared within 72 h of challenge.

Tolerance of BMNV to ether, NaCl concentration and pH was investigated by means of the infection method of the author using larval and post-larval kuruma shrimp, Penaeus japonicus. Test shrimp sampled on day 4 after water-borne inoculation were examined for nuclear hypertrophy of the mid-gut gland epithelial cells in fresh squash preparations under the dark field microscope. The results obtained are summerized as follows;Ether : BMNV did not tolerate to 18 hr treatment with ethyl ether at 4°C.NaCl concentration : BMNV was inactivated in 25% NaCl solution within 10 hours and in 12.5% NaCl solution within 24 hours. But it tolerated to 24 hr treatment with NaCl solutions of 0-6.0%.pH : BMNV had low telerance to low pH, and was inactivated in sea water adjusted to pH value of 1.0 within 10 minutes, 1.5 and 2.0 within 30 minutes, 2.5 within 60 minutes, and 3.0 and 4.0 within 180 minutes. Whereas the virus had high tolerance to high pH, and was not inactivated by 180 minute exposure in sea water adjusted to pH values of 10.0-13.0.

This paper describes the survival of BMNV in tissues of frozen or freeze-dried infected post-larval kuruma shrimp, Penaeus japonicus, and in sea water at different temperatures.Larval and post-larval kuruma shrimp were used as test animals for infectivity trials. Test animals sampled on day 4 after water-borne inoculation were examined for nuclear hypertrophy of the mid-gut gland epithelial cells in fresh squash preparations under the dark field microscope.BMNV in frozen tissues did not tolerate to 5 years storage if stored at -20°C, whereas it survived more than 7 years at high levels of infectivity if stored at -80°C. The virus in freeze-dried tissues tolerated to 7 year storage at 4°C, but having 99.9% loss of infectivity compared to that of the virus in frozen tissues stored at -80°C.BMNV in sea water tended to reduce its survival period with storage temperature rising, and was inactivated within 4 days at 30&eg;C, 7 days at 25°C, 12 days at 20°C and 20 days at 15°C.